There are two error-prone DNA polymerases of the Y-type polymerase family that have been studied extensively in E. coli: DNA polymerase IV (Pol IV) (Kenyon et al., Proc Natl Acad Sci USA 77(5), 2819-2823 (1980)) and DNA polymerase V (Pol V) (Kato et al., Molecular & General Genetics 156(2), 121-131 (1977); Steinborn et al., Molecular & General Genetics 165(1), 87-93 (1978)). Pol IV is part of the translesional DNA synthesis (TLS) pathway that allows for DNA damage repair. Pol IV greatly increases mutational activity in E. coli (Kim et al., Proc Natl Acad Sci USA 94(25), 13792-7 (1997)) and P. aeruginosa (Sanders et al., Journal of Bacteriology 188(24), 8573-8585 (2006)) when over-expressed. In fact, E. coli over-expressing Pol IV exhibits up to 200-fold higher mutational rates than cells not over-expressing Pol IV (Tompkins et al., J. Bacteriology 185(11), 3469-72 (2003)). More recently Pol IV has been shown to be lethal when over-expressed at very high levels (approximately 20 times more Pol IV than control cells), indicating that controlling the level of Pol IV is crucial to obtaining mutated genotypes (Uchida et al., Molecular Microbiology (2008)). There are approximately 250 copies of Pol IV in a single, unstressed E. coli cell (Kim et al., Molecular Genetics and Genomics 266(2), 207-215 (2001)).
The level of Pol V in unstressed E. coli is far lower than for Pol IV: approximately 5 copies per cell (Fuchs et al., DNA Repair and Replication 69, 229-264 (2004)). Pol V is encoded by UmuDC (Fuchs et al., DNA Repair and Replication 69, 229-264 (2004)). The role of Pol V is perhaps less clear than for Pol IV, but it is clear that Pol V also functions as part of the TLS system (Schlacher et al., Chemical Reviews 106(2), 406-419 (2006)). Pol V has a requirement for ATP and damaged DNA in order to be functional (Schlacher et al., Chemical Reviews 106(2), 406-419 (2006)) and is active in repairing DNA damage caused by complete replication blocks: either from UV light, polycyclic hydrocarbons or other mutagenic agents (Fuchs et al., DNA Repair and Replication 69, 229-264 (2004)). In order to replicate damaged DNA, Pol V requires the active form of RecA protein to be present (Schlacher et al., Chemical Reviews 106(2), 406-419 (2006)). Pol V requires RecA for its own activation both transcriptionally and post-transcriptionally as well as during DNA replication (Fujii et al., J. Molecular Biology 341(2), 405-17 (2004)).
Both Pol IV and Pol V are implicated in the SOS response of E. coli. As part of that system the error-prone polymerases have requirements for the β clamp subunit of the replicative polymerase Pol III (Burnouf et al., J. Molecular Biology 335(5), 1187-1197 (2004)). Without the β clamp, neither polymerase can push replication beyond a stalled replication fork (Fujii et al., J. Molecular Biology 341(2), 405-417 (2004b); Wagner et al., Embo Reports 1(6), 484-488 (2000)). However, both Pol IV and Pol V are able to facilitate DNA replication in vitro without the β clamp (Fujii et al., Journal of Molecular Biology 341(2), 405-417 (2004); Kim et al., Proc Natl Acad Sci USA 94(25), 13792-7 (1997)). Pol IV is controlled at least partially by the sigma factor RpoS in E. coli (Layton et al., Molecular Microbiology 50(2), 549-61 (2003)).
Both Pol IV [aka DinB] and Pol V have been linked to high non-targeted mutation rates in several species. Over-expression of Pol IV (or Pol V) in P. aeruginosa results in a mutator phenotype that can confer resistance to bactericidal agents.
The technique of directed evolution has been established as a method for developing bacterial and yeast strains capable of degrading recalcitrant compounds such as polyaromatic hydrocarbons and chlorinated solvents (Ang et al., Abstracts of Papers of the American Chemical Society 229, U223-U223 (2005a); Ang et al., Enzyme and Microbial Technology 37(5), 487-496 (2005b); Diaz et al., Microbiology and Molecular Biology Reviews 65(4), 523 (2001); Furukawa, Bioscience Biotechnology and Biochemistry 70(10), 2335-2348 (2006); Urlacher et al., Applied Microbiology and Biotechnology 64(3), 317-325 (2004)). Typically the technique involves the mutation of specific genes and the subsequent evolution of those genes in chemostat culture under low levels of selective pressure from the contaminant of interest or with the contaminant of interest as sole carbon source. This approach though is cumbersome and slow.